Fluorescent cell viability counter

ABSTRACT

Method and apparatus for automatically ascertaining the number of dead cells in a batch. All of the cells in the batch are stained with a specific fluorescent dye in a reservoir, then the cells are passed through a capillary tube by means of a peristaltic pump under a microscope while illuminated by an ultraviolet light. The microscope is coupled to a photomultiplier which in turn has its output applied to an electronic counter with a readout. After the dead cells are counted, the batch is immersed in a heating bath in which all the viable cells are killed and another count is obtained by repassing the batch beneath the microscope. The difference in the counts is the number of original viable cells while the first count is the original number of dead cells.

United States Patent [72] inventors lrwinJ- Kati! 3,413,464 11/1968Kamentsky 250/833 UVX 1 E851 SL1 Brooklyn, 11234; 3,497,690 2/l970Wheeless, Jr. et al. 250/71 Nicholas M. Satriano, 6410 Fitchett St., RPrimary Exammer-Arch1e R. Borchelt egoPark,N.Y.ll374,Morr1sA. A h ds S LB A l b d Benjaminson, 626 Riverside Drive, New g jy. g PP 6 York, N.Y.10031 21 Appl. No. 11,628 [22] Filed Feb. 16, 1970 [45] Patented June22, 1971 ABSTRACT: Method and apparatus for automatically ascer- E [54]g gsf figz ABILITY COUNTER taining the number of dead cells in a batch.All of the cells in mg 1g.

m the batch are stained w1th a speclfic fluorescent dye in a reser- U.S.v UV, voir then the cells are passed through a capillary tube 250/71250/71-5 23/2303 means of a peristaltic pump under a microscope whileillu- {51] Int. Cl. v0ln 21/22, minated by an umaviolet light Themicroscope is coupled to a 21/26 photomultiplier which in turn has itsoutput applied to an elec- 0 Search tronic counter a readouL After thedead ceIls are 7135 UV; 356/35 39; 424/7; 23/230 B counted, the batch isimmersed in a heating bath in which all 56 R f Ci ed the viable cellsare killed and another count is obtained by l 1 e erences t repassingthe batch beneath the microscope. The difference in UNITED STATESPATENTS the counts is the number of original viable cells while thefirst 3,327,l l9 6/ 1967 Kamentsky 250/833 UV count is the originalnumber of dead cells.

Pl/070Ml/1. 7/126 l)? ruse Z? 106/: Am? [L66 red/ tau/V761? Alva new?our 3 I? i M/(KDSMPE l9 .50L/0 $4 7M l t 1' 6 E a 2485 4 77: PumpggNpEMSER {215 M P445776 54/2 W457: RecEPMcLE =O=kk i gm/e PATENTEDJUN22 um mmbk FLUORESQENT CELL VIABILITY COUNTER STATEMENT OF GOVERNMENTINTEREST The invention described herein may be manufactured and used byor for the Government of the United States of America for governmentalpurposes without the payment of any royalties thereon or therefor.

BACKGROUND OF THE INVENTION Field of the Invention The present inventionrelates to a system for counting and discriminating between living anddead cells and more/particularly pertains to staining all the cells witha specific fluorescent dye and automatically counting the dead cells,and thereafter killing the viable cells and then counting all the cells,providing a rapid count of each.

Description of the Prior Art In the field of counting and discriminatingdead and viable cells, it has been the general practice to employ astandard technique of visually and manually counting the cells exposedto trypan blue. This method is both tedious and extremely timeconsuming. A more recent method contemplates the use of an automaticcell spectrophotometer on cells which have been stained with trypanblue. This instrument is based upon the principle of light absorptionand scattering which requires highly complex instrumentation. Thetechnique itself is less sensitive than other techniques. Such prior artdevices have been unsatisfactory in that errors are introduced byundissolved dye particulates, fluids in which the cells are suspended"and in counting small size cells. Erythrocytes must be eliminated sincethey refract light and do not take up trypan blue, thereby resulting ina false count of the living cells.

SUMMARY OF THE INVENTION The general purpose of this invention is toprovide a method and apparatus that has all the advantages of similarlyemployed prior art devices and has none of the above describeddisadvantages. To attain this, the present invention provides a uniquetechnique in which all the cells are stained with 4 acetamido 4'isothiocyanostilbene 2,2 disulfonic acid Disodium (SITS) a fluorescentdye and then passed through a capillary, illuminated by an ultravioletlight, beneath a microscope. The microscope is connected to aphotomultiplier which provides a count only for those cells whichfluoresce and applies to to a counter. All the cells are then killed byheat and recounted, thus giving, first, a count of the nonviable cellsand, thereafter, a total count of all the cells, whereby an accuraterapid count of both viable and nonviable cells is provided.

An object of the present invention is to provide a simple, accurate,reliable inexpensive and automatic device for ascertaining a count ofboth dead and living cells in a specimen.

Another object is to provide a method and technique which is direct,simple and automatic for counting both viable and nonviable cells.

Other objects and many of the attendant advantages of this inventionwill be readily appreciated as the same becomes better understood byreference to the following detailed description when considered inconnection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWING The FIGURE is a block diagram of anembodiment made in accordance with the principle of this invention andillustrates the method employed.

DESCRIPTION OF THE PREFERRED EMBODIMENT In the illustrated embodiment, asmall specimen reservoir 10 is provided at one end of the flow tube 11,which is disposed directly above a larger reservoir 12 whose inner wall13 converges downwardly toward a small opening 14 at the base and isconnected to a transparent capillary tube 15. The capillary tube ll5,which is transparent to fluorescent light, and the reservoir 12 aresupported by a clear plastic slide 16. In order to maintain balance andprevent distortion, a solid plastic block 17 is seated above thecapillary tube. The inner diameter of tube 15 is selected for theparticular size of the cells to be counted. It serves to restrict themultiple simultaneous passage of the cells therethrough in order topermit observation of the cells individually. The capillary tube extendsdirectly between the objective 18'of a standard fluorescent microscopel9 and the light condenser 20. The eyepiece end 21 of the microscope isconnected to a photomultiplier tube 22 in any of the standard methods sothat the multiplier tube directly senses the ultraviolet light fromsource 20' impinging on the eyepiece 21 and provides an output count foreach light burst detected at the microscope. The electric pulse from themultiplier is applied to an electronic counter and computer 23 whichalso includes an output indication as by a printout. This type ofcounter/computer is well known in the art and is commercially available.

The capillary 15 extends beyond the plastic block 17 and is connected toa circulating means or a peristaltic pump 24 via a flexible coupling 25while a second flexible coupling 26 connects the opposite end of thepump to a valve diverter 27. The flexible couplings decouple the pumpmovement and vibrations from the flow system. This form of pump preventsdamage or destruction of the cells while supplying the necessary motiveforce to circulate the cells. The valve 27 either directs the flow intothe waste receptacle 28 or through the tube 29 to a heating module 30.Within the module is a solution 31 which is heated by element 32connected to a source of electrical power 33 and the module may includea thermostat 34. The flow tube 35 is coiled within the heating module topermit the heating action to act on the flowing cells. After the heatingmodule, the cells are directed back to the reservoir along flow tube 11.

Having fully described the apparatus employed, the process or techniquewill now be detailed. It has been discovered that 4 acetamido 4isothiocyanostilbene 2, 2 disulfonic acid Disodium (SITS), a fluorescentdye, has the ability to distinguish between living and nonliving cells.Viable cells take up the dye and retain it in their vesicles and do notfluoresce brightly if at all, while nonviable cells fluoresce verybrightly, since the dye escapes from their vesicles. This dye is not pHdependent, has an emission spectrum of 4 1 5420 millimicrons and anactivation spectrum of 345360 millimicrons. Five milligrams of the dyeare added to each milliliter of a buffered physiological saline solutionto form a 5mg./ml. solution which is added to the unknown cellsuspension. In the above concentration the dye (SITS) is nontoxic.

The cell suspension in the dye solution is placed in the specimenreservoir 10, the ultraviolet source 20' activated and the pump 24started. When the cell suspension solution reaches flow through thecapillary tube 15 and passes beneath the objective 18 of the ultravioletmicroscope w, the photomultiplier 22 will respond only to those cellswith a high fluorescent output and provide a count at computer 23 onlyof the dead or nonviable cells per unit volume.

After all the dead cells are counted, the solution proceeds into theheating module 30, where all the viable cells are killed, resulting intheir releasing the SITS from their vesicles and providingbrightfluorescent staining. The solution then is recirculated to thespecimen reservoir I0 and held until all the initial or originalcontents therein have been depleted.

With all the cells now dead, the solution is again recirculated andcounted as previously described. This count is of the total number ofcells initially in solution. The computer 23 is also programmed tosubtract the number of nonviable cells counted in the first passage fromthe total number of cells counted in the second passage so as to printout the following information:

1. number of viable cells 2. number of nonviable cells 3. total numberof cells Bypass or diverting valve 27 is opened to the waste receptacle28 and the specimen solution discarded by being pumped therein. Thesystem is then flushed by adding washing fluid to the specimen reservoirand circulating as necessary and is thereafter discarded into the wastereceptacle.

The method of this invention comprises the following steps:

a. applying the specimen cells to be counted to a saline solution of afluorescent dye SlTS;

b. passing the cell solution through a restricted passage;

c. exposing said cell solution in said passage to an ultraviolet light;

d. detecting and counting the fluorescent light pulses thereof;

e. killing any viable cells in said solution; and then f. detecting andrecounting said fluorescent pulses.

The above described invention is not limited as to the cell type whichmay be counted or examined and the dye is specific for biologicalmaterial, and the problem of false counts is eliminated by the excellentsolubility of the dye. It should be understood, of course, that theforegoing disclosure relates to only a preferred embodiment of theinvention and that numerous modifications or alterations may be madetherein without departing from the spirit and the scope of the inventionas set forth in the appended claims.

We claim:

1. An apparatus for determining the number of viable and nonviable cellsin a mg./ml. saline solution of 4 acetamido 440 isothiocyanostilbene 2,2disulfonic acid Disodium which comprises:

an elongated restricted transparent passageway having an open end andconnected at its other end to a circulating means for flowing a liquidthrough said passageway, the opposite end of said circulating meansbeing connected to means for killing said viable cells;

coupling means connecting said killing means to said open end of saidpassageway;

a fluorescent microscope having an objective disposed on one side ofsaid passageway;

a source of ultraviolet light disposed on the opposite side of saidpassageway from said objective for directing ultraviolet light throughsaid passageway into said objective;

light amplifying and detecting means connected to said microscope fordetecting light pulses therefrom and converting them to electric signalsand, computer means connected to said detecting means for counting saidlight pulses; and

whereby when the solution is circulated twice through said apparatus acount of the number of nonviable and the total number of cells will beobtained.

2. The apparatus according to claim 1 wherein said restricted passagewayis a capillary tube.

3. The apparatus according to claim 2 wherein said circulating means isa peristaltic pump.

4. The apparatus according to claim 3 wherein said means for killing isa heating module through which the cells are passed.

5. The apparatus according to claim 4 wherein said amplifying anddetecting means is a photomultiplier circuit and tube.

6. The apparatus according to claim 5 further including a specimenreservoir disposed in line with and proximate said open end of saidpassageway.

7. A method for ascertaining the number of viable and nonviable cellswithin a specimen which comprises the steps of:

preparing a solution of said specimen cells with a saline solution of anontoxic fluorescent dye;

passing said prepared solution through a restricted passage exposingsaid prepared solution in said passage to an u

1. An apparatus for determining the number of viable and nonviable cellsin a 5mg./ml. saline solution of 4 acetamido 440 isothiocyanostilbene2,2 disulfonic acid Disodium which comprises: an elongated restrictedtransparent passageway having an open end and connected at its other endto a circulating means for flowing a liquid through said passageway, theopposite end of said circulating means being connected to means forkilling said viable cells; coupling means connecting said killing meansto said open end of said passageway; a fluorescent microscope having anobjective disposed on one side of said passageway; a source ofultraviolet light disposed on the opposite side of said passageway fromsaid objective for directing ultraviolet light through said passagewayinto said objective; light amplifying and detecting means connected tosaid microscope for detecting light pulses therefrom and converting themto electric signals and, computer means connected to said detectingmeans for counting said light pulses; and whereby when the solution iscirculated twice through said apparatus a count oF the number ofnonviable and the total number of cells will be obtained.
 2. Theapparatus according to claim 1 wherein said restricted passageway is acapillary tube.
 3. The apparatus according to claim 2 wherein saidcirculating means is a peristaltic pump.
 4. The apparatus according toclaim 3 wherein said means for killing is a heating module through whichthe cells are passed.
 5. The apparatus according to claim 4 wherein saidamplifying and detecting means is a photomultiplier circuit and tube. 6.The apparatus according to claim 5 further including a specimenreservoir disposed in line with and proximate said open end of saidpassageway.
 7. A method for ascertaining the number of viable andnonviable cells within a specimen which comprises the steps of:preparing a solution of said specimen cells with a saline solution of anontoxic fluorescent dye; passing said prepared solution through arestricted passage; exposing said prepared solution in said passage toan ultraviolet light; detecting and counting those cells whichfluoresce; killing any viable cells in said prepared solution; andrepassing said prepared solution after killing said viable cells throughsaid passage and detecting and counting the fluorescent cells.
 8. Themethod according to claim 7 wherein said dye is 4 acetamido 4''isothiocyanostilbene 2, 2 disulfonic acid Disodium.
 9. The methodaccording to claim 8 wherein said prepared solution consists of saidspecimen cells added to 5 mg. of said dye for every ml. of salinesolution.